U.S. patent number 5,603,931 [Application Number 08/289,881] was granted by the patent office on 1997-02-18 for method for delivering a bioactive molecule to a cellular target.
This patent grant is currently assigned to Boston Biomedical Research Institute. Invention is credited to Victor A. Raso.
United States Patent |
5,603,931 |
Raso |
February 18, 1997 |
**Please see images for:
( Certificate of Correction ) ** |
Method for delivering a bioactive molecule to a cellular target
Abstract
Hybrid reagents comprising a first portion having an affinity
for a cellular target and a second portion having an affinity for a
bioactive molecule are described, said hybrid reagents being
capable of selectively releasing the bioactive molecule in response
to a change in pH. The hybrid reagents of the present invention can
be used diagnostically or therapeutically.
Inventors: |
Raso; Victor A. (Brookline,
MA) |
Assignee: |
Boston Biomedical Research
Institute (Boston, MA)
|
Family
ID: |
23914241 |
Appl.
No.: |
08/289,881 |
Filed: |
August 12, 1994 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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998754 |
Dec 28, 1992 |
5501854 |
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482001 |
Feb 16, 1990 |
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Current U.S.
Class: |
424/136.1;
424/143.1; 424/150.1; 435/188; 435/70.21; 530/387.3; 530/391.1;
530/391.3; 530/391.7 |
Current CPC
Class: |
C07K
16/1045 (20130101); C07K 16/12 (20130101); C07K
16/2881 (20130101); C07K 16/468 (20130101); A61K
38/00 (20130101); C07K 2317/77 (20130101) |
Current International
Class: |
C07K
16/08 (20060101); C07K 16/46 (20060101); C07K
16/10 (20060101); C07K 16/28 (20060101); C07K
16/18 (20060101); C07K 16/12 (20060101); A61K
38/00 (20060101); C07K 016/24 (); A61K
039/395 () |
Field of
Search: |
;424/136.1,143.1,150.1
;530/391.1-4,387.3 ;435/188,70.21 |
Other References
Waldmann Science vol. 252:1657, 1991. .
Harris et al. TibTeh vol. 11, 1993, p. 42. .
Hird et al. Genes and Caneer Carney et al. ed. p. 183
1990..
|
Primary Examiner: Feisee; Lila
Attorney, Agent or Firm: Hamilton, Brook, Smith &
Reynolds, P.C.
Government Interests
GOVERNMENT FUNDING
The invention described herein was supported in whole or in part by
the National Institutes of Health. The United States Government has
certain rights to this invention.
Parent Case Text
The application is a division of application Ser. No. 07/998,754
filed Dec. 28, 1992, now U.S. Pat. No. 5,501,854 which is a
continuation of application Ser. No. 07/482,001 filed Feb. 16, 1990
now abandoned.
Claims
I claim:
1. A method for delivering a bioactive molecule having an antibody
binding site that is sensitive to pH to a cellular target within
cells of a host, comprising:
a) providing a first portion of a hybrid reagent which, upon
administration to said cells at physiologic pH, binds to the
surface of said cells with the subsequent pinching off of the
surface of said cells to form endosomes having a second and lower
pH and containing said hybrid reagent whereby the hybrid reagent is
transported to the interior of said cells by endocytosis, said
first portion comprising an antibody or antigen binding fragment
thereof; and,
b) screening antibodies or antigen binding fragments thereof which
bind the bioactive molecule to determine which of said antibodies
or antigen binding fragments bind the bioactive molecule at
physiologic pH and release it at said second and lower pH found
within the endosome;
c) selecting an antibody or antigen binding fragment thereof to
provide a second portion of said hybrid reagent which binds said
bioactive molecule at physiologic pH and releases it at said second
and lower pH; and
d) constructing a hybrid reagent containing said first portion and
said second portion;
e) administering said hybrid reagent to said host
whereby said constructed hybrid reagent binds to the surface of
said cells, is endocytosed into said cells and releases the
bioactive molecule to the cellular target with in said cells.
2. A method of claim 1 wherein said first pH is in the range of
from about 6.5 to about 7.5.
3. A method of claim 2 wherein said second and lower pH is in the
range of from about 4.5 to about 5.5.
4. A method of claim 1 wherein said bioactive molecule is a toxin,
an enzyme, a drug or a metal.
5. A method of claim 1 wherein said bioactive molecule is
diphtheria toxin or a cytotoxic mutant or cytotoxic fragment
thereof.
6. A method of claim 5 wherein said first portion comprises an
antibody or antigen binding fragment thereof specific for
transferrin receptor.
7. A method of claim 6 wherein said first pH is in the range of
from about 6.5 to about 7.5 and said second and lower pH is in the
range of from about 4.5 to about 5.5.
8. A method of claim 1 wherein said hybrid reagent is combined with
a pharmaceutically acceptable carrier.
9. A method of claim 1 wherein said first portion binds to a cell
surface receptor.
10. A method of claim 9 wherein said cell surface receptor is a
tumor-associated receptor.
11. A method of claim 9 wherein said cell surface receptor is a
viral-associated receptor.
12. A method for delivering a bioactive molecule having an antibody
binding site that is sensitive to pH to a cellular target within
the cells of a host, comprising:
a) providing a first portion of a hybrid reagent which delivers and
binds to the surface of said cells at a first pH, with the
subsequent pinching off of the surface of said cells to form
endosomes having a second and lower pH and containing said hybrid
reagent whereby the hybrid reagent is transported to the interior
of said cells by endocytosis, said first portion comprising an
antibody or antigen binding fragment thereof; and,
b) screening antibodies or antigen binding fragments thereof which
bind the bioactive molecule to determine which of said antibodies
or antigen binding fragments bind the bioactive molecule at said
first pH and release it at said second and lower pH within the
endosome;
c) selecting an antibody or antigen binding fragment thereof to
provide a second portion of said hybrid reagent which binds said
bioactive molecule at said first pH and releases it at said second
and lower pH; and
d) constructing a hybrid reagent containing said first portion and
said second portion;
e) administering said hybrid reagent to said host
whereby said constructed hybrid reagent with the antibody or
antigen binding fragment bound to the bioactive molecule binds to
the surface of said cells, is endocytosed into said cells and
releases the bioactive molecule to the cellular target with in said
cells.
Description
BACKGROUND OF THE INVENTION
Hybrid antibodies are antibodies or aggregates of antibodies which
are specific for two different antigens. Hybrid antibodies can
comprise a single antibody or fragment having a bispecific antigen
binding region (two different variable regions) or aggregates of
two or more antibodies of different specificities.
Different methods of preparing hybrid antibodies have been
reported. Auditore-Hargreaves teaches processes for preparing
hybrid antibodies by generating "half molecules" from two parent
antibodies and subsequently associating different half molecules.
See U.S. Pat. Nos. 4,470,925 (1984) and 4,479,895 (1984). Using
this process, various hybrid antibodies were prepared with
specificities for horseradish peroxidase, glucose oxidase and
theophylline.
Reading describes production of antibodies having binding
specificities for two desired antigens using a quadroma cell or a
trioma cell. See U.S. Pat. No. 4,474,893 (1984). The quadroma cell
is the fusion product of two different hybridoma cells, each of
which produce an antibody with a different specificity. A trioma
cell is the fusion product of a hybridoma and a lymphocyte which
produces antibodies with two different binding specificities.
Segal et al. describe target specific crosslinked heteroantibodies
which are used as cytotoxic agents in U.S. Pat. No. 4,676,980
(1987). Staerz et al. (1986), PNAS, 83:1453-1457, teach the use of
a hybrid antibody that can focus effective T cell activity and
Milstein et al. (1983), Nature, 305:537-539, describe the use of
hybrid antibodies in immunohistochemistry.
Raso et al., Cancer Research, 41:2073-2078 (1981) disclose the use
of hybrid antibodies with dual specificity for the plant toxin,
ricin, and immunoglobulin-bearing target cells. The hybrid
antibodies were constructed in vitro and the attachment of the
hybrid antibody-ricin complex to the human target cells was
observed using fluorescein labeled antibodies. Upon binding, the
human target cells were selectively killed by the hybrid-delivered
toxin.
Prior to the use of hybrid antibodies, chemical crosslinking or
nonspecific absorption methods were used to couple drugs and/or
toxins to antibody carriers. These agents possess certain
limitations due to the nature of the linkage. The linkage may alter
the drug or toxin such that the therapeutic or toxic activity is
reduced. Moreover, cleavage of the covalent bond may be
rate-limiting for the action of toxin inside the cell.
The use of hybrid antibodies obviated some of the problems
encountered with chemical crosslinking or non-specific absorption
methods; however, new problems were created. Because the drug or
toxin is bound to an antibody, the therapeutic or toxic activity is
generally inhibited. Hybrid antibody-delivered toxins or drugs are
inactive when bound to the antibody and only become active upon
release. However, the hybrid antibodies currently available have no
mechanism for releasing the toxin or drug from the respective
antibody binding region when the hybrid antibody reaches the target
site or the interior of the cell. Instead, they rely on fortuitous
dissociation. As a result, relatively large quantities of hybrid
antibodies containing drugs or toxins must be administered, because
only a small amount of the drug or toxin will dissociate and become
active.
SUMMARY OF THE INVENTION
This invention pertains to hybrid reagents comprising a first
portion having an affinity for a cellular target (e.g., antibody,
virus, ligand, receptor or molecule) and a second portion having an
affinity for a bioactive molecule (e.g., a toxin, drug, enzyme or
metal). The hybrid reagents can be administered in vivo where the
bind to the external surface of a cell. Once bound to the cell,
receptor-mediated endocytosis serves to pinch off the surface of
the cell forming an endosome, which has a lower pH than either
outside or within the rest of the cell. In response to the pH
change inside the endosome, the hybrid reagents of the present
invention selectively release the bioactive molecule. Once
released, the bioactive molecule is free to perform its
function.
Therefore, a major advantage of hybrid reagents of this invention
over currently available hybrid antibodies, which rely on
fortuitous dissociation of bioactive molecules, is that less of the
hybrid and bioactive molecule need to be administered to produce
the desired diagnostic or therapeutic effect.
The present invention also encompasses pharmaceutical compositions
comprising said hybrid reagents having a bioactive molecule bound
thereto, methods of immunotherapy and a method for selecting
antibodies or fragments thereof capable of binding a bioactive
molecule at one pH and releasing that molecule in response to a
change in pH.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic diagram depicting the delivery to a cell of a
bioactive molecule from a hybrid reagent by receptor mediated
endocytosis and release of the bioactive molecule in response to
the lower pH found within a cellular endosome.
FIG. 2 is a graph plotting the percent dissociation (i.e., release)
of monoclonal antibody 6B3 from diptheria toxin over 100 minutes
time at a pH of 4.5 and temperatures of 22.degree. C. and
37.degree. C.
FIG. 3 is a graph plotting the percent dissociation of monoclonal
antibody 6B3 from diptheria toxin over 30 minutes time at pH 5.0
and pH 4.5 at 37.degree. C.
FIG. 4 is a graph plotting the percent dissociation of monoclonal
antibodies 5A7 and 1F3 from diphtheria toxin over 60 minutes time
at pH 5.0 at 37.degree. C.
FIG. 5 is a graph plotting the percent incorporation of .sup.3 H
leucine over 180 minutes time as a measure of protein synthesis
inactivation by native diphtheria toxin and hybrid-delivered CRM107
in H-meso cells.
FIG. 6 is a graph plotting the toxicity dose-response curve for
hybrids and conjugates incubated for 16 hrs. with transferrin
receptor positive CEM cells.
FIG. 7 is a graph plotting the toxicity dose-response curve for HIV
and transferrin receptor directed hybrids on HIV-infected 8E5
cells.
DETAILED DESCRIPTION OF THE INVENTION
The hybrid reagents of this invention comprise a first portion
having an affinity for a cellular target and a second portion
having an affinity for a bioactive molecule (e.g., a toxin, drug,
metal or an enzyme). The hybrid reagents can be administered in
vivo where they bind to the external surface of a cell. Once bound
to the cell, receptor-mediated endocytosis serves to pinch off the
surface of the cell forming an endosome, Which has a lower pH than
either outside or within the rest of the cell. In response to the
change in pH within the endosome, the hybrid reagents selectively
release the bioactive molecule. The first portion of the hybrid can
be, for example, a ligand (e.g., transport proteins such as
transferrin, interleukin-2, LDL), a growth factor (e.g., EGF,
PDGF), an antibody, a hormone, a receptor molecule (e.g.,
recombinant CD4), a virus, or a fragment thereof and the second
portion is an antibody or an antibody fragment.
The first portion of the hybrid reagent has an affinity-for a
cellular target, such as an antigenic or receptor site on the
surface or inside a cell (i.e., a cell surface antigen or cell
surface receptor). Examples of cellular targets are Ig, common
acute lymphoblastic leukemia antigen (CALLA), B1, gp26, Ia,
transferrin receptor, EBV transformation antigen and the receptors
for ligands such as interleukin-2, MSH, insulin, thyroglobulin,
LHRH and NGF. Viral proteins on the surface of infected cells
(e.g., HIV-infected T-lymphocyte) can also serve as targets for
antibody and receptor guided hybrid reagents.
The second portion of the hybrid reagent is an antibody or antibody
fragment chat has an affinity for a bioactive molecule at one pH
and releases the bioactive molecule in response to a change in pH.
This bonding and release may be due to a number of mechanisms. For
example, the second portion of the hybrid reagent may have an
affinity for a bioactive molecule that undergoes a conformational
change in response to a change in pH. Such molecules can be
identified by using physical or other methods known in the art
(e.g., circular dichroism, fluorescence). As another example, the
second portion of the hybrid reagent may ionically bond to a
bioactive molecule at one pH and the ionic bond may break in
response to a change in pH.
A method for isolating antibodies that dissociate from molecules in
response to a change in pH is described in detail in Example 1. In
general, antibodies against a bioactive molecule are prepared using
known techniques. Clone supernatants are then assayed for the
ability to bind the molecule at the first selected pH. Clones
testing positive for binding ability are screened to isolate those
that release the molecule at a second selected pH. For example,
antibodies that bind a bioactive molecule at physiologic pH (pH
about 6.5 to 7.5) can be tested to isolate those clones that
release the molecules at acidic pH (pH less than 6.5).
Examples of bioactive molecules are plant or bacterial toxins,
drugs, enzymes and metals. Examples of useful toxins are diphtheria
toxin, pseudomonas exotoxin, ricin, pokeweed antiviral peptide
(PAP), and tricathecum. The toxins can also be genetically or
chemically altered or mutated such as CRM107 (Laird J. Virol.,
19:220-227 (1976)) and HA48DT and HA51DT (Myers et al., J. Biol.
Chem., 263:17122-17127 (1988)). Drugs which can be used in the
invention are for example, interferon, insulin, and methotrexate.
Examples of metals which can be used in the invention are
radiometals (e.g., Tc-99m, In-111, Cu-67, Pd-109, Pd-103, Re-188,
Au-198, Au-199, Ru-97, Hg-197, Ag-111, Bi-212, Os-191 and Pb-203)
and non-radioactive metals (e.g., zinc).
FIG. 1 illustrates receptor-mediated endocytosis of a hybrid
reagent-molecule complex. The first portion of the hybrid reagent
binds to the external surface of the cell, which becomes pinched
off to form an endosome. Endosomes have a pH lower than (e.g., pH
about 4.5-5.5) the pH either outside or within the rest of the cell
(e.g., pH about 6.5-7.5) (Geisow, M. L. and W. H. Evans, Ext. Cell
Res., 150:36-46 (1984)). Therefore, by using a hybrid reagent in
which the first portion has an affinity for a cell surface
component and the second portion has an affinity for a bioactive
molecule at physiologic pH and dissociates from the bioactive
molecule in response to acidic pH, a molecule can be delivered into
a cell and released within acidic compartments of cells, such as
cell endosomes.
The hybrid reagents can be produced by joining together the first
and second portions using known techniques (e.g., chemical
coupling, cell fusion, or genetic engineering techniques). The
hybrid reagents are preferably made by chemically coupling the two
portions together. For example, a disulfide linkage using
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) as the
crosslinking agent can be used (Raso et al., NATO Advanced Studies
Institute, 82:119-138 (1984)). Both portions become sparingly
substituted with pyridyldisulfide groups which are reduced to
thiols on one of the portions. Upon mixing of the two portions, the
free thiols on one of the portions readily reacts with the
unreduced groups on the second portion and form disulfide linkages.
The resulting hybrids can then be purified using gel
filtration.
When the first and second portions of the hybrid reagent are both
antibodies, two whole parental antibodies may be joined together to
produce the hybrid reagent (i.e., hybrid antibody). A variety of
crosslinking agents, such as protein A, carbodiimide, and
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) can be used to
link the whole parental antibodies (Kranz et al., Proc. Natl. Acad.
Sci. USA, 78:587 (1981); U.S. Pat. No. 4,474,893)).
The hybrid antibodies can also be produced by chemically joining
parental antibody fragments containing a sufficient portion of the
antigen binding region to allow the fragment to bind to its
respective antigen (Nisonoff et al., Arch. Biochem. Biophys.,
93:460-467 (1961) and Raso et al., Cancer Research, 41:2073-2078
(1981)). The two types of parental antibodies (i.e., one type will
become the first portion of the hybrid antibody and the other type
will become the second portion) can then be separately digested
with pepsin. Bivalent F(ab').sub.2 molecules are obtained after a
separation step such as chromatography. Equal amounts of the
2F(ab').sub.2 types can then be mixed and after reducing their
inter-heavy chain disulfide linkages, the resulting Fab' fragments
are allowed to randomly reassemble into F(ab').sub.2 dimers with
dual specificity. The dual specificities of the hybrid product can
be verified using cell-based and solid phase assays which use
radioactive or fluorescent probes (Raso, V., Immunol. Reviews,
62:93-117 (1982)).
Alternatively, the intrinsic disulfide links of the F(ab').sub.2
molecules can be reduced to thiois and the vicinal thiols generated
can be stabilized (e.g., with sodium arsenite). Ellman's reagent
can be used to activate the vicinal thiols on one type of the Fab'
fragments. Upon mixture of the reduced Fab' fragment with an
activated Fab' fragment, an exclusively bi-specific hybrid will be
formed (Brennan, M., et al., Science, 228:81-83 (1985)).
The hybrid antibodies can also be produced using cell fusion
techniques as described in U.S. Pat. No. 4,474,893, to Reading. In
this technique, hybridoma cells which secrete the parental
antibodies are fused together to form quadroma or trioma cells.
These quadroma and trioma cells secrete bi-specific antibodies
possessing the antigen binding regions of both parental
antibodies.
In addition, the hybrid antibodies can be produced using genetic
engineering techniques. In these procedures, DNA encoding the heavy
and light chain variable regions of each of the parental antibodies
are introduced into an appropriate host cell, preferably a lymphoid
cell (e.g., a myeloma cell). The transformed cell can then
synthesize, assemble and secrete the hybrid antibody.
The parental antibodies used to produce the hybrid antibody can be
selected from those presently available or can be specially
prepared. The parental antibodies can be obtained using
conventional monoclonal antibody methodology, (e.g., the standard
somatic cell hybridization techniques of Kohler and Milstein,
Nature, 256:495 (1975)).
Suitable antibodies which are specific towards tumor associated
antigens and are therefore appropriate to comprise the first
portion of the hybrid reagent, are for example, 7D3, directed
against the human transferrin receptor, (Griffin et al., Cancer
Res., 47:4266 (1987)); C19, directed against the carcinoembryonic
antigen, (Griffin et al., J. Biol Resp. Modif., 1:194 (1982));
260F9, directed against a breast cancer antigen, (Bjorn et al.,
Cancer Res., 45:1214 (1985)); 96.5 directed against a melanoma
associated antigen, (Casellas et al., In J. Cancer, 30:437 (1982));
45-2D9, directed against an oncogene product, (Roth et al., J.
Immunol., 136:2305 (1986)) and J-5, directed against the common
acute lyphoblastic leukemia antigen, (Raso et al., Cancer Res.,
42:457 (1982)).
Suitable antibodies which are specific towards diptheria toxin and
are capable of releasing the toxin in response to a change in pH
from physiologic to acidic, are D5E8, D1F3, D3E1, D6B3, D5D5, D1D5,
D5F5 and D4B7. These antibodies are therefore appropriate to
comprise the second portion of the hybrid reagent.
The hybrid reagents described herein can be used diagnostically.
For example, hybrid molecules comprising a first portion which has
an affinity for a tumor cell and a second portion which has an
affinity for a radiometal can be used to deposit radiometal within
tumor cells and thereby obtain a scintographic image of the
tumor.
Hybrid reagents of this invention can also be used therapeutically.
For example, hybrid molecules comprising a first portion having an
affinity for a viral-associated antigen (e.g., an HIV antigen) or a
viral-associated receptor and a second portion having an affinity
for a bioactive molecule, can be used therapeutically to kill or
otherwise modify virus infected cells. Similarly, hybrid molecules
comprising a first portion having an affinity for a
tumor-associated antigen or a tumor-associated receptor and a
second portion having an affinity for a bioactive molecule can be
used therapeutically to kill or otherwise modify tumor cells.
When the hybrid reagent described herein is used in a
pharmaceutical composition, it can be administered by a wide
variety of techniques. For example, intravenously, parenterally,
transdermally subcutaneously or via an implanted reservoir
containing the hybrid molecule. The form in which the hybrid
molecule will be administered (e.g., solution, emulsion) will
depend on the route by which it is administered. The quantity of
the hybrid molecule to be administered will be determined on an
individual basis and will be based at least in part on
consideration of the individual's size, the severity of the
symptoms to be treated, and the result sought.
This invention is further illustrated by the following
examples.
EXAMPLE 1
The Isolation of Anti-Diphtheria Toxin Antibodies Capable of
Releasing a Molecule at a Selected pH
Mice were immunized with progressively increasing doses of active
diphtheria toxin (1 .mu.g-3 .mu.g I.P.) or a high dose of
formalin-inactivated diphtheria toxoid (100 .mu.g I.P.). Following
a booster injection of the immunogen, spleens were removed and
fused with NS-1 cells to generate hybridomas (Kohler and Milstein,
Nature, 256:495 (1975)). Supernatants from microtiter wells with
clones were assayed for the ability to bind .sup.125 I-diphtheria
toxin using a polyethylene glycol precipitation method. Antibody
positive supernatants usually bound 25,000 cpm while negatives and
controls bound only 4,000 cpm. In a typical fusion approximately 35
positive clones were obtained from the spleen of a single
animal.
A second assay was developed in order to examine the influence of
pH on the interaction between diphtheria toxin and the different
monoclonal antibodies. Diphtheria toxin (100 .mu.l at 300 .mu.g/ml)
was absorbed to polyvinyl microtiter wells, excell was washed off
with PBS. Antibody (100 .mu.l at 1-50 .mu.g/ml) was then added,
allowed to react for two hours and the plate was washed with PBS.
Attached antibody was revealed by subsequent addition of a .sup.125
I-goat antimouse IgG reagent (background was approximately 100 cpm,
positive clones bound approximately 1,000-3,000 cpm).
To test for pH effects on toxin release, the antibody was allowed
to bind to the immobilized diphtheria toxin for two hours in
replicate wells and then a small volume of concentrated buffer was
added to provide a final pH of 7.0, 5.0 or 4.5. Dissociation was
allowed to proceed for different time intervals (5-90 minutes) at
either 23.degree. C. or 37.degree. C. (normal body temperature).
Released antibody was quickly washed off the plates with PBS and
the amount remaining was quantified using a .sup.125 I-goat
antimouse IgG probe. This method was used to identify 23 clones
producing antibody which rapidly dissociated from diphtheria toxin
at a pH of 4.5 and eight clones having antibody that was sensitive
to release at a pH of 5.0. No release occurred at a pH of 7.0.
The time-course of dissociation at pH 4.5 for one of these
monoclonal antibodies (D6B3) is shown in FIG. 2. At 23.degree. C.
the rate of release was slower and less complete than at 37.degree.
C. Approximately 80 percent of the antibody initially bound
dissociated from diphtheria toxin and most of this occurred within
the first 5 minutes. It is known that the diphtheria toxin remains
attached to the assay plate since binding of monoclonal antibodies
derived from different clones remained completely unaffected by the
same acid conditions.
FIG. 3 shows that the binding interaction of this D6B3 antibody was
much less sensitive to release at pH 5.0, with only 25 percent
having dissociated by 30 minutes in contrast to 80 percent at pH
4.5. The kinetics of release for two monoclonal antibodies which
did dissociate at pH 5.0, at 37.degree. C. is shown in FIG. 4. The
binding interaction between D5A7 and diphtheria toxin was even
disrupted at pH levels as high as 5.5. Thus a substantial fraction
of diphtheria toxin was rapidly relinquished by these different
antibodies at the precise pH and temperature conditions found in
endosomal vesicles and other acidic compartments within cells
(Geisow, J. L. and W. H. Evans, Exp. Cell Res., 150:36-46
(1984)).
The pH-dependent break-up of antibody and toxin was shown to be
based upon conformational changes in the toxin. Thus, the t.sub.
1/2 .apprxeq.1-2 min for the acid triggered dissociation of
antibody and toxin is close to the t.sub. 1/2 =30 sec for the
pH-induced transition of free toxin (Blewitt, M. G., et al.,
Biochem., 24:5458-5464 (1985)). Moreover, the D6B3 antibody bound
to formalin stabilized diphtheria toxoid at pH 7.0 but did not
release when the pH was reduced to pH 4.5. Apparently, the chemical
crosslinking of toxoid prevented the pH-induced transition which
allows D6B3 to dissociate from native toxin.
EXAMPLE 2
Hybrid-Mediated Delivery of .sup.125 I-Diphtheria Toxin to
Cells
Hybrid antibodies were formed with various anti-diphtheria toxin
antibodies by linking them to anti-transferrin receptor monoclonal
antibodies by a method previously described (Raso, F., et al., NATO
Advanced Studies Institute, 82:119-138 (1984)). The dual
specificity and cell targeting capability of these hybrids was
demonstrated using .sup.125 I-diphtheria toxin (hereinafter
.sup.125 I-DT). CEM cells derived from a patient with T-cell
leukemia (Foley, G. E., et al., Cancer, 18:522-529 (1965)), which
have abundant transferrin receptor on their surface, were used as a
test line for anti-transferrin receptor/antidiphtheria toxin
hybrids and two different routes of delivery were tested. The cells
were either pre-treated with the hybrid and washed so that the
empty toxin binding sites of surface-bound hybrids could then
capture subsequently added .sup.125 I-DT; or hybrid plus .sup.125
I-DT were pre-complexed and then used as a single agent for
reaction with the cell surface transferrin receptors.
CEM cells were incubated with the components designated in Table I
for 30 minutes at 0.degree. and then washed with PBS to remove
unbound hybrid. They were then exposed to .sup.125 I-DT for 30
minutes at 0.degree., washed with PBS and counted to measure the
amount bound to cells.
The results in Table I show that cells exposed to an
anti-transferrin receptor/anti-diphtheria toxin hybrid (7D3/D1F3)
bound five times higher levels of .sup.125 I-DT than untreated
cells. This enhanced binding was receptor-specific since
preoccupying the target epitope using excess unmodified 7D3
antibody blocked hybrid attachment and subsequent .sup.125 I-DT
binding (Table I). Hybrids formed with different anti-diphtheria
toxin monoclonal antibodies (D4B7 and D5E8) showed similar toxin
binding properties (Table I).
TABLE I ______________________________________ Binding of .sup.125
I-DT to Hybrid-Coated CEM Cells Pretreatment CPM Bound
______________________________________ None 888 7D3/D1F3 Hybrid
4,381 Excess 7D3 plus 7D3/D1F3 Hybrid 973 None 556 7D3/D4B7 Hybrid
4,306 7D3/D5E8 Hybrid 5,657
______________________________________
CEM cells were treated for 1 hour at 0.degree. C. with an
equivalent amount of .sup.125 I-DT either alone in PBS or
pre-complexed at 22.degree. for 15' to hybrid at 10.sup.-8 M (Table
II). Following treatment, the cells were washed with PBS and
counted. Table II shows that significant delivery over the basal
binding levels was attained even though the concentration of
complex used to treat these cells was relatively low (10.sup.-8
M).
TABLE II ______________________________________ Delivery of
Hybrid-Complexed .sup.125 I-DT to CEM Cells Treatment CPM Bound
______________________________________ .sup.125 I-DT alone 1,649
7D3/D4B7 Hybrid - .sup.125 I-DT complex 13,116 7D3/D5E8 Hybrid -
.sup.125 I-DT complex 15,297
______________________________________
EXAMPLE 3
Plate Assay for Dual Specificity of HIV-Directed Hybrids
An anti-HIV monoclonal antibody was elicited using a synthetic
envelope protein and used to form the HIV-specific hybrid
(anti-HIV/D5E8) by coupling it to an anti-diphtheria toxin antibody
(D5E8) following a method previously described (Raso, F., et al.,
NATO Advanced Studies Institute, 82:119-138 (1984)). A solid-phase
radioimmunoassay was devised by adsorbing the envelope peptide
antigen to the wells of polyvinyl microtitre plates. PBS and either
antibody or hybrid at 6.times.10.sup.-9 M was then added to the
well for 2 hrs, and any unbound reagent was washed off using PBS.
The dual specificity of the hybrid was demonstrated after allowing
it to bind to the coated plate via its HIV-specific combining sites
and then revealing its presence by binding .sup.125 I-CRM107 to the
free toxin-specific sites of the composite molecule. Table III
shows that the anti-HIV/D5E8 hybrid bound .sup.125 I-CRM107 while
anti-HIV alone bound no toxin even though it was attached to the
plate as evidenced by using an .sup.125 I-goat anti-mouse IgG
probe.
TABLE III ______________________________________ Plate Assay to
Demonstrate the Binding of Anti-HIV Antibody and Hybrid Amount
Bound (CPM) .sup.125 I-CRM107 .sup.125 I-G/M
______________________________________ PBS 307 253 anti-HIV 112
1,501 anti-HIV/D5E8 Hybrid 1,245 --
______________________________________
EXAMPLE 4
Hybrid-Mediated Cytotoxicity of a Mutated Form of Diphtheria
Toxin
The availability of genetically or chemically altered diphtheria
toxin cogeners (e.g., CRM107) with no capacity for attaching to
cells provides an added dimension to the hybrid delivery approach.
The cell-binding defect which makes these analogs non-toxic to
cells can be restored via the hybrid carrier moiety so that its
lethal action is aimed exclusively at the selected cell surface
target.
Human mesothelioma cells (H-Meso) were used to test the
effectiveness of anti-transferrin receptor/anti-diphtheria toxin
hybrids (7D3/D1F3 and 7D3/D5E8) for restoring the full cytotoxic
potential of CRM107. The H-meso cells were incubated for 2 hours at
37.degree. C. with 4.times.10.sup.-8 M CRM107 alone;
(4.times.10.sup.-8 M) CRM107 in combination with the hybrids
7D3/D5E8 or 7D3/D1F3 at 1.times.10.sup.-8 M, or 4.times.10.sup.-8 M
CRM107 in combination with the hybrids (1.times.10.sup.-8 M) plus
excess anti-receptor antibody (7D3) (10.sup.-5 M). Cells were then
pulse labeled with .sup.3 H-leucine for 30 min. H-meso cells in
media to which 10 mM NH.sub.4 Cl was added were also incubated with
the same components.
The data in Table IV show that while CRM107 alone was incapable of
entering cells and inhibiting protein synthesis, it became a very
potent and rapid-acting cytotoxin when used in combination with the
hybrid antibodies. This lethal action was dependent upon
hybrid-mediated delivery to transferrin receptors since little
toxicity was obtained when these sites were blocked by including an
excess of free anti-receptor antibody (7D3) during the 2 hour
incubation time (Table IV).
The acid environment of intracellular compartments is essential for
cytotoxicity since this induces the release of CRM107 from the
antibody and translocation into the cytosol where it inactivates
elongation factor 2. This condition was demonstrated by adding
NH.sub.4 Cl to the cells. This weak base, which is known to raise
vesicle pH, greatly reduced the ability of the hybrid-CRM107
combination to kill H-Meso cells (Table IV). The same experiments
were carried out using the anti-HIV/D5E8 hybrid (2.times.10.sup.-8
M) plus CRM107 (4.times.10.sup.-8 M) using HIV-infected 8E5 cells
as the target (Folks, T. M., et al., J. Exp. Med., 164:280-290
(1986)). The same acid-dependency was demonstrated (Table V).
TABLE IV ______________________________________ Hybrid-Mediated
Cytotoxicity of CRM107 Tested on Human Mesothelioma Cells (2-hr
Assay); Transferrin Receptor Specificity and Acid-Dependency .sup.3
H-Leucine Incorporation Inhibition (CPM) (Percent)
______________________________________ H-Meso Cells 92,560 --
+CRM107 90,755 2 +7D3/D5E8 + CRM107 1,605 98 +7D3/D1F3 + CRM107
8,050 91 +excess 7D3 + 7D3/D5E8 + 52,325 43 CRM107 +excess 7D3 +
7D3/D1F3 + 53,960 42 CRM107 H-Meso Cells + 10 mM NH.sub.4 Cl 92,435
-- +CRM107 76,885 17 +7D3/D5E8 + CRM107 52,105 44 +7D3/D1F3 +
CRM107 80,802 13 ______________________________________
TABLE V ______________________________________ Acid-Dependency of
Hybrid-Mediated Cytotoxicity of CRM107 on HIV-Positive 8E5 Cells
.sup.3 H-Leucine Incorporation Inhibition (CPM) (percent)
______________________________________ HIV-Positive + 8E5 cells
alone 103,955 -- +CRM107 85,140 18 +NH.sub.4 Cl + CRM107 82,115 21
+anti-HIV/D5E8 + CRM107 21,820 79 +NH.sub.4 Cl + anti-HIV/D5E8 +
78,985 24 CRM107 ______________________________________
The transferrin receptor directed hybrid-CRM107 complex was assayed
on human colon adenocarcinoma cells to determine if the same high
cytotoxic potency found for the HoMeso and HIV-infected 8E5 cell
lines extended to alternative malignant cell types. The combined
action of CRM107 plus hybrid at 10.sup.-8 M produced extensive cell
kill within two hours and its potency was comparable to 10.sup.-7 M
native diphtheria toxin (Table VI). These results indicate that
hybrid-delivery not only renders CRM107 cytotoxic to cells but also
suggests that its entry via the transferrin pathway is as efficient
as diphtheria toxin uptake by its usual mechanism. Moreover, a
transferrin/D5E8 conjugate was constructed to examine if
transferrin itself would mediate delivery of CRM107 into cells. In
fact, this natural ligand coupled to the anti-diphtheria toxin
monoclonal antibody (D5E8) provided a similar level of toxicity as
the anti-transferrin receptor (7D3) guided hybrid.
TABLE VI ______________________________________ Lethal Effects of
Anti-Transferrin Receptor Directed Hybrid Plus CRM107 on Human
Colon Adenocarcinoma Cells (2-hr. Assay) .sup.3 H-Leucine
Incorporation Inhibition (CPM) (Percent)
______________________________________ LS174T cells 54,070 --
+CRM107 (10.sup.-7 M) 48,355 11 +7D3/D5E8 (10.sup.-8 M) + 1,930 96
CRM107 (10.sup.-7 M) +Diphtheria Toxin (10.sup.-7 M) 1,785 97
+Diphtheria Toxin (10.sup.-8 M) 6,295 88
______________________________________
In addition to using the transferrin receptor as a target for
hybrid delivery, the common acute lymphoblastic leukemia antigen
(CALLA) was similarly tested as a site of entry into CALLA-bearing
Nalm-1 leukemia cells (Raso, V., et al., Cancer Res., 42:457-464
(1982)). An anti-CALLA/D5E8 hybrid was formed and examined for its
ability to kill these cells in combination with CRM107 following
the protocol set forth for H-meso cells and anti-transferrin
receptor/anti-diphtheria toxin. However, incubation was carried out
for 6 hours at the same temperature (Table VII).
Good cell kill was achieved by targeting the hybrid-CRM107 to this
distinct membrane site; however, the longer incubation time
required suggests that entry and/or release of toxin was slower
than for transferrin receptor directed agents.
TABLE VII ______________________________________ CALLA-Directed
Cytotoxic Action of Hybrid-CRM107 on Nalm 1 Cells (6 hr. Assay)
.sup.3 H-Leucine Incorporated Inhibition (CPM) (Percent)
______________________________________ Nalm-1 Cells 22,130 --
+anti-CALLA/D5E8 24,110 0 +CRM107 22,820 0 +anti-CALLA/D5E8 + 4,080
82 CRM107 ______________________________________
EXAMPLE 5
Kinetics of Cytotoxicity in H-Meso Cells
One of the fundamental premises underlying the acid-triggered
hybrid carrier concept predicts that this mode of delivery will not
interfere with the normal mechanism of toxin action after specific
targeting has been achieved. A critical measure of toxin efficiency
can be obtained by monitoring the kinetics of inhibition of protein
synthesis. This parameter accurately indicates how rapidly toxin
gains access to its target in the cytosol (e.g., elongation factor
2) and was therefore used to evaluate hybrid-delivered CRM107 (FIG.
5).
H-Meso cells were incubated at 37.degree. C. for the designated
intervals with either 10.sup.-8 M diphtheria toxin, 10.sup.-8 M
CRM107, or the anti-transferrin receptor/anti-diphtheria toxin
hybrid (7D3/D3E1)-CRM107 combination at 10.sup.-8 M. The cells were
then pulse labeled with .sup.3 H-leucine for 30 minutes to measure
the extent incorporation into protein compared to untreated control
cells. The time course of protein synthesis inhibition as reflected
by .sup.3 H-leucine incorporation, for H-Meso cells incubated with
10.sup.-8 M diptheria toxin alone, 10.sup.-8 M CRM107 alone or with
the anti-transferrin receptor/anti-diphtheria toxin hybrid
(7D3/D3E1) plus CRM107 at 10.sup.-8 M was then measured.
FIG. 5 shows that both native toxin and the CRM107 hybrid
combination gave identical kinetics profiles which were
characterized by a 30-40 minute lag period followed by a rapid
inactivation phase with t.sub. 1/2 =24 minutes and t.sub. 1/2 =26
minutes respectively. Unbound CRM107 alone at 10.sup.-8 M had no
effect on the ability of the cells to synthesize protein. The fact
that hybrid-delivered CRM107 killed cells as fast as native
diphtheria toxin suggests that its release from the antibody
combining site was unimpeded and that there was no interruption of
the normal course of events required for its lethal action.
Finally, a covalently-coupled anti-transferrin receptor-CRM107
conjugate (7D3-CRM107) was constructed by standard
disulfide-linkage methods and its cytotoxic effect compared with
the effect produced by the 7D3/D5E8 hybrid plus CRM107. Transferrin
receptor positive CEM cells were incubated for 16 hours at
37.degree. C. with the designated concentrations of the 7D3/D5E8
hybrid plus 10.sup.-7 M CRM107, the 7D3-CRM107 disulfide-linked
covalent conjugate and native diphtheria toxin, CRM107 alone or
7D3/D5E8 hybrid alone. The cells were then pulse labeled with
.sup.3 H-leucine for 30 minutes and the amount of incorporation
into protein was compared with untreated control cells.
FIG. 6 shows the toxicity dose response curves of the hybrid, the
conjugate and native diphtheria toxin. The conjugate, 7D3-CRM107
was cytotoxic to transferrin receptor positive cells, the kinetics
of cell killing was much slower than that found for
hybrid-delivered CRM107. CEM cells are not particularly sensitive
to diphtheria toxin as reflected in the ID.sub.50
=2.times.10.sup.-9 M obtained with native toxin. The
transferrin-receptor directed 7D3-CRM107 conjugate was slightly
more effective, giving an ID.sub.50 =1.times.10.sup.-9 M. In
contrast, hybrid-delivered CRM107 (ID.sub.50 =4.times.10.sup.-12 M)
was 250-fold more potent than the covalent conjugate, based upon
the concentration of hybrid added. Neither the 7D3/D5E8 hybrid
alone nor CRM107 alone had an effect upon the cells. These results
indicate that covalent coupling can impede toxin action since the
disulfide-linked 7D3-CRM107 conjugate was slower acting and less
potent than the corresponding 7D3/D5E8 hybrid delivered CRM107.
FIG. 7 shows dose response curves for inhibition of protein
synthesis in HIV-infected 8E5 cells after 16 hr exposure to CRM107
plus hybrids directed against either HIV or transferrin receptors
on the cell membrane. The ID.sub.50 for the anti-HIV/D5E8 hybrid
plus CRM107 was 2.times.10.sup.-9 M but this reagent became
10-times more potent when nicked CRM107 (cleaved at a specific site
using trypsin) was used (ID.sub.50 =2.times.10.sup.-10 M). It is
believed that proteolytic cleavage is a prerequisite for toxic
activity and normally occurs at the cell surface or in subcellular
compartments. This anti-HIV hybrid-mediated cytotoxicity was
blocked by neutralizing intracellular compartments with NH.sub.4 Cl
(Table V) and the uninfected control cell line was not affected by
hybrid-delivered CRM107.
Equivalents
Those skilled in the art will know, or be able to ascertain using
no more than routine experimentation, many equivalents to the
specific embodiments of the invention as described herein. These
and all other equivalents are intended to be encompassed by the
following claims.
* * * * *